Chloride transport in normal and cystic fibrosis epithelial cells

The experimental work described in this thesis was aimed at a better understanding of the pathogenesis of CF with the emphasis on electrophysiological studies on Cl" transport. Chloride transport was studied with Ussing chamber, isotopic ('25I")-efflux and fluorescent (SPQ) measurements as well as with the patch-damp technique. In search for a suitable cell culturing model, we studied C!" transport in CF keratinocytes, which are easy to obtain and would provide us with sufficient cell material (Chapter 3). However, the use of keratinocytes appeared to be limited to primary cell cultures which can only be passaged for maximal 10 times, necessatating the use of many different and often heterogeneous N and CF cell lines necessary. The availibility of a continuously growing cell line with CF genotype and phenotype would provide a more homogeneous model for biochemical, electrophysiological and genetic studies. We have therefore immortalized a CF nasal polyp cell line with a hybrid SV40/ Ad12 virus, and have characterized this cell line biochemically and electrophysiologically as discussed in Chapter 4. A continuously growing homogeneous cell line selected for its chloride secreting characteristics would also he of much interest for CF research. The subclone 19A of the human colon carcinoma cell line HI29 is such a cell line, easy to maintain in culture without the multiple addition;; to the medium required for culturing keratinocytes and primary nasal polyp cells and without the use of feeder cells. These colonocytes can be coru;idered as a model for the study of Cl· transport in normal cells. In Chapter 5 we describe the patchclamp experiments with HI29.ci19A cells performed to characterize the cAMPdependent regulation of the outwardly rectifYing chloride channel which was thought to be defectively regulated in CF. Apart from cAMP regulation of chloride channels, we also found that G-proteiru; are involved in the regulation of a different type of chloride channel in intestinal membranes. Chapter 6 describes the vesicle and patch-damp experiments leading to the identification of a novel potential signal transduction pathway for chloride transport in intestinal cells. The homology between CFIR and different types of pump proteins like MDR led us to verify the hypothesis that CFIR could function as a pump with substrates exerting secondary effects on chloride channels. In Chapter 7 MDR-substrates and -blockers were tested on epithelial chloride traru;port and on the outwardly rectifYing chloride channel in particular.